Acute effects of haemodialysis on circulating microparticles

O R I G I N A L A R T I C L E

Acute effects of haemodialysis on circulating microparticles

Philip de Laval ¹ , Fariborz Mobarrez ² , Tora Almquist ³ , Liina Vassil ¹ , Bengt Fellstro¨m ¹ and Inga Soveri ¹

1 Department of Medical Sciences, Uppsala University, Uppsala, Sweden, ² Department of Medicine, Karolinska Institutet, Unit of Rheumatology, Karolinska University Hospital, Solna, Sweden and ³ Division of Nephrology, Department of Clinical Sciences, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden

Correspondence and offprint requests to: Philip de Laval; E-mail: philip.de.laval@medsci.uu.se

ABSTRACT

Background. Microparticles (MPs) are small cell membrane-derived vesicles regarded as both biomarkers and mediators of biological effects. Elevated levels of MPs have previously been associated with endothelial dysfunction and predict cardiovascular death in patients with end-stage renal disease. The objective of this study was to measure change in MP concentrations in contemporary haemodialysis (HD).

Methods. Blood was sampled from 20 consecutive HD patients before and 1 h into the HD session. MPs were measured by flow cytometry and phenotyped based on surface markers.

Results. Concentrations of platelet (CD41

) (P ¼ 0.039), endothelial (CD62E

) (P ¼ 0.004) and monocyte-derived MPs (CD14

) (P < 0.001) significantly increased during HD. Similarly, endothelial- (P ¼ 0.007) and monocyte-derived MPs (P ¼ 0.001) expressing tissue factor (TF) significantly increased as well as MPs expressing Klotho (P ¼ 0.003) and receptor for advanced glycation end products (RAGE) (P ¼ 0.009). Furthermore, MPs expressing platelet activation markers P-selectin (P ¼ 0.009) and CD40L (P ¼ 0.045) also significantly increased. The increase of endothelial (P ¼ 0.034), monocyte (P ¼ 0.014) and RAGE

MPs (P ¼ 0.032) as well as TF

platelet-derived MPs (P ¼ 0.043) was significantly higher in patients treated with low-flux compared with high-flux dialysers.

Conclusion. Dialysis triggers release of MPs of various origins with marked differences between high-flux and low-flux dialysers. The MPs carry surface molecules that could possibly influence coagulation, inflammation, oxidative stress and endothelial dysfunction. The clinical impact of these findings remains to be established in future studies.

Keywords: chronic kidney disease, haemodialysis, Klotho, microparticles, RAGE

INTRODUCTION

Patients with chronic kidney disease (CKD) have high incidence of cardiovascular disease (CVD), and CVD remains as the major cause of mortality in patients treated with haemodialysis (HD).

In addition to traditional risk factors, the increased risk is

attributed to non-traditional risk factors, including inflamma- tion, endothelial dysfunction and oxidative stress [1]. Plasma concentration of membrane microparticles (MPs) has been shown to be associated with cardiovascular risk [2] and patient outcome following myocardial infarction [3].

Received: 8.5.2018; Editorial decision: 1.10.2018

V

The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/

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456 doi: 10.1093/ckj/sfy109

Advance Access Publication Date: 30 October 2018 Original Article

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MPs are small vesicles, 0.1–1.0 mm in size, formed by the out- ward blebbing of the cell membrane, released from cells upon activation or due to apoptosis and necrosis [4]. Although plate- let-derived MPs (PMPs) are most abundant in plasma, MPs from various cell types have been observed, including MPs of endo- thelial (EMPs) and monocyte (MMPs) origin [5]. MPs retain char- acteristics of their cell of origin such as cytosolic content and membrane antigens that determine their function and can be used for identification purposes [4]. Today, MPs are accepted as both biomarkers and bioactive particles involved in various dis- ease states [5]. Most MPs express phosphatidylserine (PS) on their outer membrane and some express tissue factor (TF), which in effect makes MPs procoagulant [6]. PS facilitates bind- ing of coagulation cascade proteins Factor (F)VII, IX, X and prothrombin and the assembly of coagulation complexes, and TF is considered the prime activator of the coagulation cas- cade [7]. In addition to PS and TF, PMPs may also express markers of platelet activation including P-selectin (CD62P) [6]

and CD40L [8].

Klotho is a transmembrane protein found predominantly in the distal convoluted tubule of the kidney. Klotho is a co-factor that makes the fibroblast growth factor (FGF) receptor specific for FGF-23, which lowers renal phosphate resorption and sup- presses synthesis of calcitriol [1, 25(OH)

D

] in the kidney [9].

Klotho expression declines with reducing renal function and Klotho deficiency has been speculated to contribute to vascular calcification in CKD [10].

Advanced glycation end products (AGEs) are elevated in CKD not only due to hyperglycaemia but also due to oxidative stress. AGEs can reciprocally induce oxidative stress and in- flammation and have been linked to CVD [11]. HD decreases the level of AGEs [12]. The receptor for AGEs (RAGE) is expressed by various cells including endothelial cells and mac- rophages and is upregulated in diabetes and inflammatory dis- eases [11].

Previous studies on the acute effects of HD on levels of circu- lating MPs are scarce and to our knowledge, no previous studies have examined Klotho or RAGE expression in MPs. In this study, we investigated MP formation in contemporary HD by measur- ing plasma concentrations of a wide range of MP subtypes be- fore and during HD.

MATERIALS AND METHODS

Subjects

Twenty patients on thrice weekly maintenance HD at the Department of Nephrology at Uppsala University Hospital who consented to participate in the study were included. There were no other exclusion criteria. At study start, all patients at the di- alysis units were asked to participate. Inclusion was stopped as soon as 20 patients meeting the inclusion criteria had given their consent. The study was conducted in accordance with the Declaration of Helsinki. All participants provided written in- formed consent, and the Regional Ethics Review Board in Uppsala approved the trial.

Sampling

The patients received their prescribed dialysis with no modifi- cations made for study purpose. For each patient, 2 mL of blood was drawn from the venous line and collected in citrate vacu- tainer tubes. Samples were taken before the start of the dialysis session and 1 h into dialysis. The blood was centrifuged within

30 min of collection at 1500g for 10 min at room temperature (RT) in order to obtain platelet poor plasma (PPP). The plasma was frozen and stored at –70

C directly.

Measurement of MPs

PPP was thawed in a water bath for 5 min at 37

C and subse- quently centrifuged at RT for 20 min at 2000g, in order to further remove any debris or cells that may interfere with the analysis.

The supernatant was then re-centrifuged at 13 000g for 2 min at RT. Subsequently, 20 mL of the supernatant were incubated for 20 min in the dark with phalloidin-Alexa 660 (cell-fragment marker, Invitrogen, Paisley, UK) [13], lactadherin-Fluorescein isothiocyanate (FITC) (Haematologic Technologies, VT, USA).

For detection of MP origin either CD41-PE (Beckman Coulter, Brea, CA, USA) for PMPs, CD62E-APC (AH diagnostics, Stockholm, Sweden) for EMPs or CD14-FITC (Beckman Coulter, Brea, CA, USA) for MMPs was added. In addition, exposure of TF (CD142-PE, BD, NJ, USA) was measured on PMPs, MMPs and EMPs; CD40L (CD154-APC, AH diagnostics) and P-selectin (CD62P-APC, AH diagnostics) on PMPs and Klotho (Klotho-FITC, Bioss Antibodies Inc, Woburn, MA, USA) and RAGE (Anti-RAGE- FITC, Abcam, Cambridge, UK). MPs were measured using flow cytometry on a Beckman Gallios instrument (Beckman Coulter).

The MP gate was determined using Megamix beads (0.5–3.0 mm, BioCytex, Marseille, France). MPs were defined as particles

<1.0 mm in size and positive or negative to the markers de- scribed above. Conjugate isotype-matched immunoglobulins with no reactivity against human antigens were used as nega- tive controls. In this study, results are shown as numbers of MPs (MP counted  standard beads added/L)/standard beads counted (FlowCount, Beckman Coulter). The intra- and interas- say coefficients of variation for MP measurement were <9%.

Statistical analysis

All statistical analysis was performed using Rstudio (Version 1.1.383). Prior to analysis, data were log-transformed, if neces- sary, to obtain normal distribution. Histograms and QQ-plots were used to assess normality in combination with Shapiro–

Wilk test. Samples taken before and 1 h into dialysis were com- pared using paired t-test for paired samples with normally or log-normally distributed variables and Wilcoxon signed-rank test for variables with non-normal distribution. Change in MP concentration between groups was compared with Student’s t-test for normally distributed data and Mann–Whitney U test for non-normal data. Correlations between clinical parameters and changes in MP concentrations were assessed using Spearman’s rank correlation. P < 0.05 were considered significant.

RESULTS

Patient characteristics

Twenty patients were included in the study, but samples from one patient were excluded due to failure to comply with the study protocol during sampling. Patient characteristics are pre- sented in Table 1. HD was performed using synthetic dialysers.

In eight subjects, high flux polysulphone dialysers were utilized.

The rest of the patients were dialysed using polyamide/polyary- lether-sulfone/polyvinylpyrrolidone blend dialysers, with nine being low-flux and two high-flux.

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Microparticles

Plasma concentrations of the measured MPs are presented in detail in Table 2 and Figure 1. Plasma concentration of total PS

MPs did not significantly change during the first hour of HD (P ¼ 0.129) but PMPs (P ¼ 0.039), EMPs (P ¼ 0.004) and MMPs (P < 0.001) increased significantly. Similarly, Klotho

(P ¼ 0.003) and RAGE

(0.009) MPs as well as PMPs with platelet activation markers CD40L (P ¼ 0.045) and CD62P (P ¼ 0.009) increased signif- icantly. A significant increase in TF

EMPs (P ¼ 0.004) and MMPs (P ¼ 0.001) was also observed but not in TF

PMPs.

A/V fistula versus dialysis catheter

Subgroup analysis revealed a significantly higher increase in Klotho

MPs in patients with arteriovenous fistula (A/V) fistula as vascular access [605.5 (range 272–846)  10

MPs/L with A/V fistula versus 247 (762–779)  10

MPs/L with dialysis catheter, P ¼ 0.036].

Low-flux versus high-flux dialysers

Increase in EMPs (P ¼ 0.034), MMPs (P ¼ 0.014), RAGE

MPs (P ¼ 0.032), and TF

PMPs (P ¼ 0.043), TF

EMPs (P ¼ 0.027) and TF

MMPs (P ¼ 0.048) were significantly higher in patients treated with low-flux compared with high-flux dialysers (Table 3). In the low-flux group, EMPs (P ¼ 0.004), MMPs (P < 0.001), Klotho

(P ¼ 0.039) and RAGE

MPs (P ¼ 0.003), and TF

EMPs (P ¼ 0.004) and TF

MMPs (P ¼ 0.009) increased signifi- cantly during dialysis and in the high-flux group MMPs (P ¼ 0.014), Klotho

MPs (P ¼ 0.019) and P-selectin

PMPs (P ¼ 0.049) increased significantly. Patients in the low-flux group were significantly older (P ¼ 0.011), had lower body mass index (BMI) (P ¼ 0.006), and were dialysed for shorter duration (P ¼ 0.013) and with less ultrafiltration volume (P ¼ 0.028) than those in the high-flux group.

Other subpopulations

No significant difference in the change of plasma concentration was found for any of the MP subpopulations depending on gender, smoking status, previous CVD events, presence of dia- betes, current statin treatment or HD modality (haemodiafiltra- tion (HDF) versus standard HD).

Correlations

Correlations between MP levels and dialysis and patient-specific factors are presented in detail in the supplementary material (Supplementary data, Table S1). Changes in Klotho

and RAGE

MP levels correlated negatively with ultrafiltration volume and BMI. Change in RAGE

MP concentrations correlated positively

with age. Changes in levels of PS

MPs and TF

PMPs and MMPs correlated positively with pre-dialysis systolic blood pressure (BP) and changes in levels of PS

MPs, MMPs and TF

PMPs, EMPs and MMPs correlated positively with post-dialysis systolic BP.

DISCUSSION

Summary of findings

In this study, we set out to obtain a comprehensive overview of the acute effect of contemporary HD on MP levels by measuring MPs in plasma before and 1 h into dialysis. The major findings of this study are (i) plasma concentration of PMPs, EMPs and MMPs increased during HD; (ii) PMPs expressing platelet activa- tion markers P-selectin and CD40L, and TF

PMPs, TF

EMPs and TF

MMPs increased during HD; (iii) Klotho

and RAGE

MPs, which have, to our knowledge, never been demonstrated before, increased significantly during HD; and (iv) the increase of MPs was generally greater in patients treated with low-flux compared with high-flux dialysers.

Previous data on MPs in relation to HD are conflicting, which could possibly be explained by difference in inclusion criteria, dialyser type and choice of antibodies used for analysis. In par- ticular, antibodies for detection of EMPs vary greatly between studies with most utilizing CD31 [14–18], and others CD66E [19], CD144 [15, 19, 20] and CD146 [20, 21]. In this study, E-selectin was used, which is upregulated on endothelial cells as an in- flammatory response. Lactadherin was used to label PS

MPs, which binds more selectively than the more commonly used Annexin V [13].

In agreement with our findings, some previous studies have reported an increase in PMP levels during dialysis [21, 22], whereas others have not [14, 19, 23]. EMP levels were reported to increase in one previous study [14], similar to our results, whereas two other studies did not observe any significant alter- ations [19, 21]. Only one previous study investigated MMPs, with no significant changes between before and after HD [19].

We demonstrated a significant increase in TF

EMPs and TF

MMPs but not TF

PMPs. The only previous study on the effect of HD on TF

MPs did not demonstrate a significant change in TF

MP plasma concentrations [19]. One study compared TF

Table 2. Plasma concentration of MPs during HD (10

MPs/L)

Factor Pre-dialysis 1 h into dialysis P-value All MPs

PS

3645 (1960–9784) 4388 (1966–12672) 0.129

Klotho

2260 (6276) 2612 (6414) 0.003

RAGE

154 (122–1356) 252 (178–1491) 0.009 PMPs (CD41

)

All PMPs 464 (153–2321) 774 (169–3000) 0.039

TF

349 (187–1149) 424 (258–1526) 0.242

CD40L

205 (33–992) 365 (52–1236) 0.045

P-Selectin

186 (50–1098) 550 (70–1369) 0.009 EMPs (CD62E

)

All EMPs 683 (188–1183) 764 (297–1795) 0.004

TF

135 (15–392) 171 (26–900) 0.007

MMPs (CD14

)

All MMPs 216 (175–443) 337 (205–584) <0.001

TF

31 (11–121) 58 (17–193) 0.001

Data presented as median (range) and mean (6SD).

Table 1. Patient characteristics

Age, mean 6 SD, years 74.1 6 10.1

Male, N (%) 14 (73.7)

BMI, median (range), kg/m

26.6 (21.0–40.7)

Arteriovenous fistula, N (%) 6 (31.6)

High-flux membrane, N (%) 10 (52.6)

Haemodiafiltration, N (%) 9 (47.4)

Dialysis duration, median (range), h 4 (4–5)

Ultrafiltration, mean 6 SD, L 1.82 6 1.21

Diabetes mellitus, N (%) 7 (36.8)

Previous CVD, N (%) 13 (68.4)

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MPs between HD, peritoneal dialysis, non-dialysed uraemic patients and healthy controls and found no significant differ- ence between groups [17]. However, neither of these studies di- vided TF

MPs by subpopulations which makes comparison difficult.

RAGE

MPs increased significantly during HD and to our knowledge, this is the first time RAGE

MPs have been observed.

The cellular origin of these MPs is not known, and we cannot conclude if the increase is specific to RAGE or just reflects a gen- eral increase in MPs. In the absence of disease, RAGE is expressed predominantly in lung tissue but under pathological conditions, including inflammatory diseases, RAGE is upregu- lated and found on various cells including monocytes and endo- thelial cells [24, 25]. This suggests that our findings possibly reflect an inflammatory response induced by HD. Studies on HD and RAGE are scarce, but soluble RAGE has been shown to be el- evated in HD patients compared with healthy controls [26].

However, HD has been shown to decrease the level of AGEs [12].

We observed a slight increase in Klotho

MPs during HD.

Klotho expression in MPs has to our knowledge never been

demonstrated before and interestingly, levels of Klotho

MPs were substantially higher than the other MP subtypes, except for PS

MPs. The origin of these MPs remains unknown. Klotho is predominantly expressed in the kidney, most abundantly, but not exclusively, in the distal convoluted tubule [27]. The kidney has also been shown to be the prime source of circulating Klotho in mice [27], which suggests that Klotho

MPs could also be of renal origin. Increase in Klotho

MPs was higher in sub- jects where an A/V-fistula was used as access compared with dialysis catheter. Previous studies have to our knowledge only included patients dialysed through A/V fistula [19, 21, 23, 28].

Interestingly, the increase in EMPs, MMPs, Klotho

MPs and TF

PMPs, EMPs and MMPs was higher in patients treated with low-flux dialysers compared with high-flux dialysers. However, it should be noted that this study was not specifically designed for the purpose of detecting differences between different dialy- sers. The intended advantage of high-flux membranes is the greater clearance of middle molecules, which might influence MP formation indirectly. Proteins involved in bioincompatibility reactions against biomaterials, including complement factors FIGURE 1: Boxplot of plasma concentrations of MPs (not classified by cell type) (A) and PMPs (B), EMPs (C) and MMPs (D) in HD with whiskers representing 1.5  interquartile range and circles representing outliers. *P < 0.05; **P < 0.01; ***P < 0.001.

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C3a and C5a, are significantly removed by filtration in high-flux HD [29]. In vitro data suggest that complement activation trig- gers MP release [30]. It is debated whether high-flux membranes are more bio-compatible than their low-flux counterparts [31, 32] although two multicentre studies have reported survival benefits [33, 34]. Pore size would not influence MP levels directly since the average pore sizes of the two high-flux dialysers used were 3.3 nm and 40.1 nm, far smaller than the lower size limit of MPs. It is possible that the lower MP formation observed in the high-flux group could in part be dependent on the use of HDF since it was predominant (90%) in this group. This is sup- ported by previous studies which have shown that patients treated with HDF have lower EMP levels compared with patients treated with standard HD [16], with no differences between HDF modalities [18, 22]. Other significant differences between the high- and low-flux groups included age, BMI, dialysis duration and ultrafiltration, further stressing the need for additional ade- quately powered studies specifically designed for this purpose to draw any major conclusions. Previous studies have not com- pared low- and high-flux membranes although dialysers of dif- ferent materials have been compared. No differences between polysulphone and cellulose triacetate dialysers have been shown in previous studies, nor between synthetic and celluloid dialysers [23].

Possible mechanism

The changes to MP levels observed during HD could be attrib- uted to many factors. The adverse effects of HD in triggering co- agulation, inflammation and oxidative stress are well established [35]. The inflammatory response has been attrib- uted to blood–biomaterial interactions resulting in protein adsorption on the dialysis membrane with subsequent activa- tion of the complement system [36]. Dialysers made from syn- thetic polymers such as polysulphone, most used in contemporary HD, are considered more biocompatible but their hydrophobic nature leads to greater protein adsorption, result- ing in a more indirect interaction with the blood [37]. Studies have shown that the protein complex, C5b-9, formed in the last step of the complement cascade induces release of MPs in vitro [30, 38].

The repetitive mechanical stress from the HD treatment it- self could contribute to MP elevation during HD, as high shear stress has previously been demonstrated to promote MP release from platelets [39]. Contrary to what we observed in this study, the removal of uraemic toxins during HD could theoretically suppress EMP formation. These toxins have been linked to en- dothelial dysfunction, and in vitro studies have demonstrated that the uraemic toxins p-cresol and indoxyl sulphate induce MP release from endothelial cells [21]. Considering the conflict- ing data on EMP levels in HD in previous studies, other mecha- nisms seem to be involved as well. In addition, these toxins are highly protein bound that makes removal during HD less effec- tive with high variation between different dialyser types [21].

Clinical significance

The pro-coagulatory potential of MPs is widely recognized [7]. In vitro studies have shown that MPs from uraemic patients have greater procoagulant activity than MPs from healthy control [17]. PMP count has also been shown to be significantly higher in CKD patients with a recent history of thrombotic events [23].

It is conceivable that the observed increase in TF

MPs could act as a source for the TF-dependent clotting during extracorporeal treatments [31]. The activation of platelets is also considered an important step in these clotting reactions, which could be reflected in the observed increases in MPs positive for platelet activation markers [40]. MPs have been linked to CVD and endo- thelial dysfunction [5]. In a prospective study, EMP concentra- tion at baseline was found to be an independent predictor of cardiovascular and all-cause mortality in patients’ dialysed with polysulphone and AN69 dialysers [41]. EMP count has also been found to correlate with pulse wave velocity (PWV) and ca- rotid augmentation index, markers of arterial stiffness, in HD patients, independently of age and BP [15]. Correlations were not found for total MPs or PMPs. Another study similarly dem- onstrated an independent association between EMP count and PWV in children with CKD [20]. Arterial stiffness is an indepen- dent predictor of cardiovascular and all-cause mortality in end-stage renal disease [42]. In vitro studies using rat aortas incubated in MPs from HD patients show that MPs impair endothelial function by reducing vasorelaxation in response to acetylcholine and cyclic guanosine monophosphate genera- tion [15].

Limitations

To appreciate these findings, a number of limitations need to be addressed. First, the sample size was limited and the studied patients were heterogeneous with regard to prescribed dialysis and clinical characteristics. However, these consecutive patients reflected the real-life setting. Patient and dialysis-spe- cific characteristics with the potential to influence the studied biomarkers need to be addressed in future trials. Secondly, the measured MP concentration was not corrected for alterations in haematocrit resulting from haemoconcentration induced by HD. However, the observed concentration changes occurred al- ready at 1 h into dialysis. Furthermore, total MP concentration was not altered during HD and none of the measured MPs corre- lated positively with ultrafiltration volume. Thirdly, the limited number of sample instances limits our understanding of the full dynamics of MP levels during HD. The typical HD sessions lasts for up to 5 h and it could be that these observed alterations are of a transient nature. Fourthly, the observed increase in Klotho

and RAGE

MPs has never been demonstrated before, and it is Table 3. Comparison of changes in MP concentrations (D10

MPs/L)

between high- and low-flux dialysers

Factor High flux Low flux P-value

All MPs

PS

366 (61851) 1722 (62815) 0.241

Klotho

250 (289 to 758) 617 (762 to 846) 0.053

RAGE

24 (6103) 128 (691) 0.032

PMPs

All PMPs 241 (6352) 340 (6755) 0.725

TF

42 (647 to 694) 74 (4 to 758) 0.043

CD40L

74 (6105) 143 (6301) 0.527

P-Selectin

142 (58 to 1040) 89 (82 to 1124) 0.720 EMPs

All EMPs 38 (319 to 626) 127 (95 to 804) 0.034

TF

18 (217 to 560) 61 (8 to 552) 0.027

MMPs

All MMPs 75 (678) 136 (661) 0.014

TF

24 (640) 44 (638) 0.048

Data presented as median (range) and mean (6SD).

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necessary to reproduce these results before any major conclu- sions are reached. Finally, the suggested differences in MP for- mation between HD using low-flux and high-flux dialysers may also be explained by other patient or dialysis-related factors as subjects were not randomly assigned treatments and groups were not matched.

CONCLUSION

HD triggers an acute increase in MPs of various origins and our data suggest a marked difference between high- and low-flux dialysers. Interestingly, MPs expressing Klotho and RAGE, which also increase during HD, have been demonstrated for the first time. The surface molecules found on MPs are not only biologi- cal markers but also carry out biological effects that are known to influence coagulation, inflammation, levels of oxidative stress and endothelial dysfunction. The clinical impact of MPs in HD remains to be established in future studies.

SUPPLEMENTARY DATA

Supplementary data are available at ckj online.

ACKNOWLEDGEMENTS

The authors thank Yvonne Lundholm, R.N., for sampling and skilful sample handling.

FUNDING

This study was supported by grants from CUWX Counties Renal Patients Foundation, ALF Grant Region Uppsala and Uppsala-O ¨ rebro Regional Research Council.

CONFLICT OF INTEREST STATEMENT

None declared.

REFERENCES

1. Sarnak MJ, Levey AS, Schoolwerth AC et al. Kidney disease as a risk factor for development of cardiovascular disease: a statement from the American Heart Association Councils on Kidney in Cardiovascular Disease, High Blood Pressure Research, Clinical Cardiology, and Epidemiology and Prevention. Circulation 2003; 108: 2154–2169

2. Baron M, Boulanger CM, Staels B et al. Cell-derived micropar- ticles in atherosclerosis: biomarkers and targets for pharma- cological modulation? J Cell Mol Med 2012; 16: 1365–1376 3. Morel O, Hugel B, Jesel L et al. Sustained elevated amounts of

circulating procoagulant membrane microparticles and sol- uble GPV after acute myocardial infarction in diabetes melli- tus. Thromb Haemost 2004; 91: 345–353

4. Hugel B, Martı´nez MC, Kunzelmann C. Membrane micropar- ticles: two sides of the coin. Physiology (Bethesda) 2005; 20:

22–27

5. Burger D, Schock S, Thompson CS et al. Microparticles: bio- markers and beyond. Clin Sci 2013; 124: 423–441

6. Mobarrez F, He S, Bro¨ijersen A et al. Atorvastatin reduces thrombin generation and expression of tissue factor, P- selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease. Thromb Haemost 2011; 106: 344–352

7. Owens AP, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 2011; 108: 1284–1297

8. Mobarrez F, Sjo¨vik C, Soop A et al. CD40L expression in plasma of volunteers following LPS administration: a com- parison between assay of CD40L on platelet microvesicles and soluble CD40L. Platelets 2015; 26: 486–490

9. Kuro-O M. Phosphate and Klotho. Kidney Int 2011; 79121:

S20–S23

10. Hu MC, Kuro-o M, Moe OW. The emerging role of Klotho in clinical nephrology. Nephrol Dial Transplant 2012; 27:

2650–2657

11. Stinghen AEM, Massy ZA, Vlassara H et al. Uremic toxicity of advanced glycation end products in CKD. J Am Soc Nephrol 2016; 27: 354–370

12. Floridi A, Antolini F, Galli F et al. Daily haemodialysis improves indices of protein glycation. Nephrol Dial Transplant 2002; 17: 871–878

13. Mobarrez F, Antovic J, Egberg N et al. A multicolor flow cyto- metric assay for measurement of platelet-derived micropar- ticles. Thromb Res 2010; 125: e110–e116

14. Boulanger CM, Amabile N, Gue´rin AP et al. In vivo shear stress determines circulating levels of endothelial micropar- ticles in end-stage renal sisease. Hypertension 2007; 49:

902–908

15. Amabile N, Gue´rin AP, Leroyer A et al. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 2005;

16: 3381–3388

16. Ariza F, Merino A, Carracedo J et al. Post-dilution high con- vective transport improves microinflammation and endo- thelial dysfunction independently of the technique. Blood Purif 2013; 35: 270–278

17. Gao C, Xie R, Yu C et al. Thrombotic role of blood and endo- thelial cells in uremia through phosphatidylserine exposure and microparticle release. PLoS One 2015; 10: e0142835 18. Esquivias-Motta E, Martı´n-Malo A, Buendia P et al.

Hemodiafiltration with endogenous reinfusion improved microinflammation and endothelial damage compared with online-hemodiafiltration: a hypothesis generating study.

Artif Organs 2017; 41: 88–98

19. Trappenburg MC, van Schilfgaarde M, Frerichs FCP et al.

Chronic renal failure is accompanied by endothelial activa- tion and a large increase in microparticle numbers with re- duced procoagulant capacity. Nephrol Dial Transplant 2012;

27: 1446–1453

20. Dursun I, Poyrazoglu HM, Gunduz Z et al. The relationship between circulating endothelial microparticles and arterial stiffness and atherosclerosis in children with chronic kidney disease. Nephrol Dial Transplant 2009; 24: 2511–2518

21. Faure V, Dou L, Sabatier F et al. Elevation of circulating endo- thelial microparticles in patients with chronic renal failure. J Thromb Haemost 2006; 4: 566–573

22. Sakurai K, Saito T, Yamauchi F et al. Comparison of the effects of predilution and postdilution hemodiafiltration on neutrophils, lymphocytes and platelets. J Artif Organs 2013;

16: 316–321

23. Ando M, Iwata A, Ozeki Y et al. Circulating platelet-derived microparticles with procoagulant activity may be a potential cause of thrombosis in uremic patients. Kidney Int 2002; 62:

1757–1763

24. Kierdorf K, Fritz G. RAGE regulation and signaling in inflam- mation and beyond. J Leukoc Biol 2013; 94: 55–68

25. Yan SF, Yan SD, Ramasamy R et al. Tempering the wrath of RAGE: an emerging therapeutic strategy against diabetic

Downloaded from https://academic.oup.com/ckj/article-abstract/12/3/456/5149791 by Uppsala Universitetsbibliotek user on 29 November 2019

complications, neurodegeneration and inflammation. Ann Med 2009; 41: 408–422

26. Nakashima A, Carrero JJ, Qureshi AR et al. Effect of circulat- ing soluble receptor for advanced glycation end products (sRAGE) and the proinflammatory RAGE ligand (EN-RAGE, S100A12) on mortality in hemodialysis patients. Clin J Am Soc Nephrol 2010; 5: 2213–2219

27. Olauson H, Mencke R, Hillebrands J-L et al. Tissue expression and source of circulating aKlotho. Bone 2017; 100: 19–35 28. Daniel L, Fakhouri F, Joly D et al. Increase of circulating neu-

trophil and platelet microparticles during acute vasculitis and hemodialysis. Kidney Int 2006; 69: 1416–1423

29. Jørstad S, Smeby LC, Balstad T et al. Generation and removal of anaphylatoxins during hemofiltration with five different membranes. Blood Purif 1988; 6: 325–335

30. Sims PJ, Faioni EM, Wiedmer T et al. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for co- agulation factor Va and express prothrombinase activity.

J Biol Chem 1988; 263: 18205–18212

31. Davenport A. The role of dialyzer membrane flux in bio-in- compatibility. Hemodial Int 2008; 12 (Suppl 2): S29–S33 32. Kerr PG, Huang L. Review: membranes for haemodialysis.

Nephrology (Carlton) 2010; 15: 381–385

33. Krane V, Krieter DH, Olschewski M et al. Dialyzer membrane characteristics and outcome of patients with type 2 diabetes on maintenance hemodialysis. Am J Kidney Dis 2007; 49: 267–275

34. Delmez JA, Yan G, Bailey J et al. Cerebrovascular disease in maintenance hemodialysis patients: results of the HEMO Study. Am J Kidney Dis 2006; 47: 131–138

35. Ekdahl KN, Soveri I, Hilborn J et al. Cardiovascular disease in haemodialysis: role of the intravascular innate immune sys- tem. Nat Rev Nephrol 2017; 13: 285–296

36. Nilsson B, Ekdahl KN, Mollnes TE et al. The role of comple- ment in biomaterial-induced inflammation. Mol Immunol 2007; 44: 82–94

37. Cheung AK. Biocompatibility of hemodialysis membranes.

J Am Soc Nephrol 1990; 1: 150–161

38. Moskovich O, Fishelson Z. Live cell imaging of outward and inward vesiculation induced by the complement C5b-9 com- plex. J Biol Chem 2007; 282: 29977–29986

39. Miyazaki Y, Nomura S, Miyake T et al. High shear stress can initiate both platelet aggregation and shedding of procoagu- lant containing microparticles. Blood 1996; 88: 3456–3464 40. Davenport A. What are the anticoagulation options for

intermittent hemodialysis? Nat Rev Nephrol 2011; 7:

499–508

41. Amabile N, Gue´rin AP, Tedgui A et al. Predictive value of cir- culating endothelial microparticles for cardiovascular mor- tality in end-stage renal failure: a pilot study. Nephrol Dial Transplant 2012; 27: 1873–1880

42. London GM. Cardiovascular calcifications in uremic patients: clinical impact on cardiovascular function. J Am Soc Nephrol 2003; 14: S305–S309

Downloaded from https://academic.oup.com/ckj/article-abstract/12/3/456/5149791 by Uppsala Universitetsbibliotek user on 29 November 2019